SEQU-INTO: Early detection of impurities, contamination and off-targets (ICOs) in long read/MinION sequencing.

The MinION sequencer by Oxford Nanopore Technologies turns DNA and RNA sequencing into a routine task in biology laboratories or in field research. For downstream analysis it is required to have a sufficient amount of target reads. Especially prokaryotic or bacteriophagic sequencing samples can contain a significant amount of off-target sequences in the processed sample, stemming from human DNA/RNA contamination, insufficient rRNA depletion, or remaining DNA/RNA from other organisms (e.g. host organism from bacteriophage cultivation). Such impurity, contamination and off-targets (ICOs) block read capacity, requiring to sequence deeper. In comparison to second-generation sequencing, MinION sequencing allows to reuse its chip after a (partial) run. This allows further usage of the same chip with more sample, even after adjusting the library preparation to reduce ICOs. The earlier a sample's ICOs are detected, the better the sequencing chip can be conserved for future use. Here we present sequ-into, a low-resource and user-friendly cross-platform tool to detect ICO sequences from a predefined ICO database in samples early during a MinION sequencing run. The data provided by sequ-into empowers the user to quickly take action to preserve sample material and chip capacity. sequ-into is available from https://github.com/mjoppich/sequ-into.

Tryptophan usage by Helicobacter pylori differs among strains.

Because of its association with severe gastric pathologies, including gastric cancer, Helicobacter pylori has been subject of research for more than 30 years. Its capacity to adapt and survive in the human stomach can be attributed to its genetic flexibility. Its natural competence and its capacity to turn genes on and off allows H. pylori to adapt rapidly to the changing conditions of its host. Because of its genetic variability, it is difficult to establish the uniqueness of each strain obtained from a human host. The methods considered to-date to deliver the best result for differentiation of strains are Rapid Amplification of Polymorphic DNA (RAPD), Multilocus Sequence Typing (MLST) and Whole Genome Sequencing (WGS) analysis. While RAPD analysis is cost-effective, it requires a stable genome for its reliability. MLST and WGS are optimal for strain identification, however, they require analysis of data at the bioinformatics level. Using the StainFree method, which modifies tryptophan residues on proteins using 2, 2, 2, - trichloroethanol (TCE), we observed a strain specific pattern of tryptophan in 1D acrylamide gels. In order to establish the effectiveness of tryptophan fingerprinting for strain identification, we compared the graphic analysis of tryptophan-labelled bands in the gel images with MLST results. Based on this, we find that tryptophan banding patterns can be used as an alternative method for the differentiation of H. pylori strains. Furthermore, investigating the origin for these differences, we found that H. pylori strains alters the number and/or position of tryptophan present in several proteins at the genetic code level, with most exchanges taking place in membrane- and cation-binding proteins, which could be part of a novel response of H. pylori to host adaptation.

Helicobacter pylori culture as a key tool for diagnosis in Colombia.

INTRODUCTION: The presence of H. pylori in the stomach is associated with gastric pathologies. However, its diagnosis through culture methods is challenging because of its complex nutritional requirements and microaerophilic conditions for optimal growth. The preferred method for rapid diagnosis of H. pylori is the Rapid Urease Test (RUT) from human biopsies, which relies on the high activity of the urease enzyme present in H. pylori. However, RUT cannot say much more information about H. pylori. This makes evident the need for bacterial culture to know essential information such as the strain type, the kind of infection present and the bacteria's antibiotic susceptibility. METHODOLOGY: Gastric biopsies from 347 patients were used for H. pylori isolation. We correlated the culture results with the RUT and histological grading used at Hospital Universitario Fundación SantaFe de Bogotá (HU-FSFB), Colombia. The concordance between techniques was determined by the Cohen's Kappa coefficient (K). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were also calculated. RESULTS: The culture standardization was successful, and it could be applied for diagnosis in the clinical practice. H. pylori was positive by culture in 88 (26.34%) patients. The concordance of RUT and culture was strong (K= 0.805), and between histology and culture was moderate (K= 0.763) as well as for the gold standard defined and culture (K= 0.80). CONCLUSIONS: We present evidence that RUT and histological methods will be better interpreted for diagnosis of H. pylori if combined with bacterial isolation in cholesterol enriched culture.

Genotipificación de cagA y de la región intermedia de vacA en cepas de Helicobacter pylori aisladas de pacientes adultos colombianos y su asociación con enfermedades gástricas / Genotyping of cagA and the intermediate region of vacA in strains of Helicobacter pylori isolated from Colombian adult patients and associations with gastric diseases

Resumen Objetivo: este estudio caracteriza la diversidad de los genes de virulencia cagA (gen asociado con la citotoxina A) y vacA (citotoxina vacuolizante) en pacientes colombianos para determinar posibles asociaciones entre estos 2 genes y la severidad de los hallazgos endoscópicos teniendo en cuenta todos los genotipos reportados para el gen vacA (s, m e i). Materiales y métodos: Helicobacter pylori fue detectado por cultivo y por métodos moleculares en biopsias de 62 pacientes. Los genotipos de cagA y vacA (m/i/s) se determinaron por reacción en cadena de la polimerasa (PCR) y secuenciación. Resultados: se aislaron 124 cepas de 62 pacientes; de estas, el 48,5% (n = 48) fueron vacA s2/m2/i2-cagA (-) presente en su mayoría en pacientes con gastritis folicular; mientras el 32,3% (n = 32) fueron vacA s1/m1/i1-cagA (+) presentes mayormente en pacientes con gastritis folicular, gastritis crónica y posible metaplasia. Se encontró una asociación significativa entre la presencia de cagA y el genotipo vacA s1/m1/i1 y la ausencia de cagA y el genotipo vacA s2/m2/i2 (p <0,001). No se encontró una asociación significativa entre la severidad de los hallazgos endoscópicos y el estatus cagA-vacA de las cepas. Conclusión: se encontró una baja prevalencia de cepas cagA (+), el estatus cagA-vacA no es un predictor de riesgo en la población estudiada y la presencia de infecciones heterogéneas sin tropismo sugieren la necesidad de tomar biopsias tanto del cuerpo como del antro del estómago en la práctica clínica rutinaria.

Abstract Objective: This study characterizes the diversity of cagA and vacA virulence genes in Colombian patients to determine possible associations between them and the severity of endoscopic findings. It considers all four genotypes reported for the vacA gene (s, m and i). Materials and methods: Helicobacter pylori was detected in biopsies of 62 patients through culturing and by molecular methods. Genotypes of cagA and vacA (m/i/s) were determined by PCR and sequencing. Results: One hundred twenty four strains from 62 patients were isolated. Of these, 48.5% (n = 48) were vacA s2/m2/i2 - cagA (-) which were mostly found in patients with follicular gastritis; 32.3% (n = 32) were vacA s1/m1/i1-cagA (+) which were mostly found in patients with follicular gastritis, chronic gastritis and possible metaplasia. Significant associations were found between the presence of cagA and the vacA s1/m1/i1 genotype and the absence of cagA and the vacA s2/m2/i2 genotype (p <0.001). No significant association was found between the severity of endoscopic findings and the cagA-vacA status of the strains. Conclusion: We found a low prevalence of cagA (+) strains, the cagA-vacA status is not a predictor of risk in this population. Moreover, the presence of heterogenous infections without tropism suggests a need for biopsies from both the corpus and the antrum of the stomach in routine clinical practice.

Optimized semi-quantitative blot analysis in infection assays using the Stain-Free technology.

Western blots are a commonly used method for protein detection and quantification in biological samples. Compensation of loading variations is achieved by housekeeping protein (HKP) normalization and/or total protein normalization (TPN). However, under infection conditions, HKP normalization, traditionally used in cell biology for quantification of western blots, can be problematic. Binding of microbes to target cells via specific receptors can induce signal transduction events resulting in drastic changes in the level of expression of HKPs. Additionally, samples collected after infection assays will include cellular and microbial proteins altering the analysis with TPN. Here we demonstrate under experimental infection conditions, how a reliable semi-quantitative analysis of proteins in western blots can be achieved using the Stain-Free technology.

The CagA toxin of Helicobacter pylori: abundant production but relatively low amount translocated.

CagA is one of the most studied pathogenicity factors of the bacterial pathogen Helicobacter pylori. It is injected into host cells via the H. pylori cag-Type IV secretion system. Due to its association with gastric cancer, CagA is classified as oncogenic protein. At the same time CagA represents the 4(th) most abundant protein produced by H. pylori, suggesting that high amounts of toxin are required to cause the physiological changes or damage observed in cells. We were able to quantify the injection of CagA into gastric AGS epithelial cells in vitro by the adaptation of a novel protease-based approach to remove the tightly adherent extracellular bacteria. After one hour of infection only 1.5% of the total CagA available was injected by the adherent bacteria, whereas after 3 hours 7.5% was found within the host cell. Thus, our data show that only a surprisingly small amount of the CagA available in the infection is finally injected under in vitro infection conditions.

Detection of cytoplasmic proteins from Helicobacter pylori in Colony Lift Immunoassay.

Use of the Colony Lift Immunoassay has been described for several Gram negative bacteria of medical interest. In all cases detection was limited to the use of antibodies against outer membrane proteins. Here we describe the adaptation of this method for detection of the cytoplasmic CagA toxin from Helicobacter pylori.

Yersinia enterocolitica exploits different pathways to accomplish adhesion and toxin injection into host cells.

The current paradigm suggests that Yersinia enterocolitica (Ye) adheres to host cells via the outer membrane proteins Yersinia adhesin A (YadA) or invasin (Inv) to facilitate injection of Yops by the type III secretion system. In this process Inv binds directly to ß1 integrins of host cells while YadA may bind indirectly via extracellular matrix proteins to ß1 integrins. Here we challenged this paradigm and investigated the requirements for Yop injection. We demonstrate that Inv- but not YadA-mediated adhesion depends on ß1 integrin binding and activation, and that tight adhesion is a prerequisite for Yop injection. By means of novel transgenic cell lines, shRNA approaches and RGD peptides, we found that YadA, in contrast to Inv, may use a broad host cell receptor repertoire for host cell adhesion. In the absence of ß1 integrins, YadA mediates Yop injection by interaction with αV integrins in cooperation with yet unknown cofactors expressed by epithelial cells, but not fibroblasts. Electron microscopic and flow chamber studies revealed that a defined intimate contact area between Ye and host cells resulting in adhesion forces resisting shear stress is required for Yop injection. Thus, the indirect binding of YadA to a broad extracellular matrix (ECM) binding host cell receptor repertoire of different cell types makes YadA a versatile tool to ensure Yop injection. In conclusion, given the differential expression of the outer membrane proteins Inv and YadA in the course of Ye infection and differential expression of integrins by various host cell populations, the data demonstrate that Ye is flexibly armed to accomplish Yop injection in different host cell types, a central event in its immune evasion strategy.

Dynamics of the Cag-type IV secretion system of Helicobacter pylori as studied by bacterial co-infections.

Many pathogenic Gram-negative bacteria possess type IV secretion systems (T4SS) to inject effector proteins directly into host cells to modulate cellular processes to their benefit. The human bacterial pathogen Helicobacter pylori, a major aetiological agent in the development of chronic gastritis, duodenal ulcer and gastric carcinoma, harbours the cag-T4SS to inject the cytotoxin associated Antigen (CagA) into gastric epithelial cells. This results in deregulation of major signalling cascades, actin-cytoskeletal rearrangements and eventually gastric cancer. We show here that a pre-infection with live H. pylori has a dose-dependent negative effect on the CagA translocation efficiency of a later infecting strain. This effect of the 'first' strain was independent of any of its T4SS, the vacuolating cytotoxin (VacA) or flagella. Other bacterial pathogens, e.g. pathogenic Escherichia coli, Campylobacter jejuni, Staphylococcus aureus, or commensal bacteria, such as lactobacilli, were unable to interfere with H. pylori's CagA translocation capacity in the same way. This interference was independent of the ß1 integrin receptor availability for H. pylori, but certain H. pylori outer membrane proteins, such as HopI, HopQ or AlpAB, were essential for the effect. We suggest that the specific interference mechanism induced by H. pylori represents a cellular response to restrict and control CagA translocation into a host cell to control the cellular damage.

Structural insights into Helicobacter pylori oncoprotein CagA interaction with ß1 integrin.

Infection with the gastric pathogen Helicobacter pylori is a risk factor for the development of gastric cancer. Pathogenic strains of H. pylori carry a type IV secretion system (T4SS) responsible for the injection of the oncoprotein CagA into host cells. H. pylori and its cag-T4SS exploit α5ß1 integrin as a receptor for CagA translocation. Injected CagA localizes to the inner leaflet of the host cell membrane, where it hijacks host cell signaling and induces cytoskeleton reorganization. Here we describe the crystal structure of the N-terminal ~100-kDa subdomain of CagA at 3.6 Å that unveils a unique combination of folds. The core domain of the protein consists of an extended single-layer ß-sheet stabilized by two independent helical subdomains. The core is followed by a long helix that forms a four-helix helical bundle with the C-terminal domain. Mapping of conserved regions in a set of CagA sequences identified four conserved surface-exposed patches (CSP1-4), which represent putative hot-spots for protein-protein interactions. The proximal part of the single-layer ß-sheet, covering CSP4, is involved in specific binding of CagA to the ß1 integrin, as determined by yeast two-hybrid and in vivo competition assays in H. pylori cell-culture infection studies. These data provide a structural basis for the first step of CagA internalization into host cells and suggest that CagA uses a previously undescribed mechanism to bind ß1 integrin to mediate its own translocation.

CagI is an essential component of the Helicobacter pylori Cag type IV secretion system and forms a complex with CagL.

Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma and MALT lymphoma, uses the Cag type IV secretion system to induce a strong proinflammatory response in the gastric mucosa and to inject its effector protein CagA into gastric cells. CagA translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it is considered as a major bacterial virulence trait. Recently, it has been shown that binding of the type IV secretion apparatus to integrin receptors on target cells is a crucial step in the translocation process. Several bacterial proteins, including the Cag-specific components CagL and CagI, have been involved in this interaction. Here, we have examined the localization and interactions of CagI in the bacterial cell. Since the cagI gene overlaps and is co-transcribed with the cagL gene, the role of CagI for type IV secretion system function has been difficult to assess, and conflicting results have been reported regarding its involvement in the proinflammatory response. Using a marker-free gene deletion approach and genetic complementation, we show now that CagI is an essential component of the Cag type IV secretion apparatus for both CagA translocation and interleukin-8 induction. CagI is distributed over soluble and membrane-associated pools and seems to be partly surface-exposed. Deletion of several genes encoding essential Cag components has an impact on protein levels of CagI and CagL, suggesting that both proteins require partial assembly of the secretion apparatus. Finally, we show by co-immunoprecipitation that CagI and CagL interact with each other. Taken together, our results indicate that CagI and CagL form a functional complex which is formed at a late stage of secretion apparatus assembly.

Effects of cholesterol on Helicobacter pylori growth and virulence properties in vitro.

BACKGROUND: Colonization of the gastric mucosa by Helicobacter pylori is often associated with chronic gastric pathologies in humans. Development of disease correlates with the presence of distinct bacterial pathogenicity factors, such as the cag type IV secretion system (cag-T4SS), the vacuolating cytotoxin (VacA), or the ability of the bacteria to acquire and incorporate cholesterol from human tissue. MATERIALS AND METHODS: The in vitro growth of H. pylori requires media (Brucella broth) complemented with vitamins and horse serum or cyclodextrins, prepared as blood agar plates or liquid cultures. Liquid cultures usually show a slow growth. Here, we describe the successful growth of H. pylori strains 26695, P217, P12, and 60190 on serum-free media replacing serum components or cyclodextrins with a commercially available cholesterol solution. RESULTS: The effects of cholesterol as a substitute for serum or cyclodextrin were rigorously tested for growth of H. pylori on agar plates in vitro, for its general effects on bacterial protein synthesis (the proteome level), for H. pylori's natural competence and plasmid DNA transfer, for the production of VacA, and the general function of the cag-pathogenicity island and its encoded cag-T4SS. Generally, growth of H. pylori with cholesterol instead of serum supplementation did not reveal any restrictions in the physiology and functionality of the bacteria except for strain 26695 showing a reduced growth on cholesterol media, whereas strain 60190 grew more efficient in cholesterol- versus serum-supplemented liquid medium. CONCLUSIONS: The use of cholesterol represents a considerable option to serum complementation of growth media for in vitro growth of H. pylori.

Neisseria meningitidis adhesin NadA targets beta1 integrins: functional similarity to Yersinia invasin.

Meningococci are facultative-pathogenic bacteria endowed with a set of adhesins allowing colonization of the human upper respiratory tract, leading to fulminant meningitis and septicemia. The Neisseria adhesin NadA was identified in about 50% of N. meningitidis isolates and is closely related to the Yersinia adhesin YadA, the prototype of the oligomeric coiled-coil adhesin (Oca) family. NadA is known to be involved in cell adhesion, invasion, and induction of proinflammatory cytokines. Because of the enormous diversity of neisserial cell adhesins the analysis of the specific contribution of NadA in meningococcal host interactions is limited. Therefore, we used a non-invasive Y. enterocolitica mutant as carrier to study the role of NadA in host cell interaction. NadA was shown to be efficiently produced and localized in its oligomeric form on the bacterial surface of Y. enterocolitica. Additionally, NadA mediated a ß1 integrin-dependent adherence with subsequent internalization of yersiniae by a ß1 integrin-positive cell line. Using recombinant NadA(24-210) protein and human and murine ß1 integrin-expressing cell lines we could demonstrate the role of the ß1 integrin subunit as putative receptor for NadA. Subsequent inhibition assays revealed specific interaction of NadA(24-210) with the human ß1 integrin subunit. Cumulatively, these results indicate that Y. enterocolitica is a suitable toolbox system for analysis of the adhesive properties of NadA, revealing strong evidence that ß1 integrins are important receptors for NadA. Thus, this study demonstrated for the first time a direct interaction between the Oca-family member NadA and human ß1 integrins.

Helicobacter pylori type IV secretion apparatus exploits beta1 integrin in a novel RGD-independent manner.

Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (T4SS) into host cells is a major risk factor for severe gastric diseases, including gastric cancer. However, the mechanism of translocation and the requirements from the host cell for that event are not well understood. The T4SS consists of inner- and outer membrane-spanning Cag protein complexes and a surface-located pilus. Previously an arginine-glycine-aspartate (RGD)-dependent typical integrin/ligand type interaction of CagL with alpha5beta1 integrin was reported to be essential for CagA translocation. Here we report a specific binding of the T4SS-pilus-associated components CagY and the effector protein CagA to the host cell beta1 Integrin receptor. Surface plasmon resonance measurements revealed that CagA binding to alpha5beta1 integrin is rather strong (dissociation constant, K(D) of 0.15 nM), in comparison to the reported RGD-dependent integrin/fibronectin interaction (K(D) of 15 nM). For CagA translocation the extracellular part of the beta1 integrin subunit is necessary, but not its cytoplasmic domain, nor downstream signalling via integrin-linked kinase. A set of beta1 integrin-specific monoclonal antibodies directed against various defined beta1 integrin epitopes, such as the PSI, the I-like, the EGF or the beta-tail domain, were unable to interfere with CagA translocation. However, a specific antibody (9EG7), which stabilises the open active conformation of beta1 integrin heterodimers, efficiently blocked CagA translocation. Our data support a novel model in which the cag-T4SS exploits the beta1 integrin receptor by an RGD-independent interaction that involves a conformational switch from the open (extended) to the closed (bent) conformation, to initiate effector protein translocation.

Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission.

The spread of retroviruses between cells is estimated to be 2-3 orders of magnitude more efficient when cells can physically interact with each other. The underlying mechanism is largely unknown, but transfer is believed to occur through large-surface interfaces, called virological or infectious synapses. Here, we report the direct visualization of cell-to-cell transmission of retroviruses in living cells. Our results reveal a mechanism of virus transport from infected to non-infected cells, involving thin filopodial bridges. These filopodia originate from non-infected cells and interact, through their tips, with infected cells. A strong association of the viral envelope glycoprotein (Env) in an infected cell with the receptor molecules in a target cell generates a stable bridge. Viruses then move along the outer surface of the filopodial bridge toward the target cell. Our data suggest that retroviruses spread by exploiting an inherent ability of filopodia to transport ligands from cell to cell.

Visualization of retroviral replication in living cells reveals budding into multivesicular bodies.

Retroviral assembly and budding is driven by the Gag polyprotein and requires the host-derived vacuolar protein sorting (vps) machinery. With the exception of human immunodeficiency virus (HIV)-infected macrophages, current models predict that the vps machinery is recruited by Gag to viral budding sites at the cell surface. However, here we demonstrate that HIV Gag and murine leukemia virus (MLV) Gag also drive assembly intracellularly in cell types including 293 and HeLa cells, previously believed to exclusively support budding from the plasma membrane. Using live confocal microscopy in conjunction with electron microscopy of cells generating fluorescently labeled virions or virus-like particles, we observed that these retroviruses utilize late endosomal membranes/multivesicular bodies as assembly sites, implying an endosome-based pathway for viral egress. These data suggest that retroviruses can interact with the vps sorting machinery in a more traditional sense, directly linked to the mechanism by which cellular proteins are sorted into multivesicular endosomes.